Upregulation of COX4-2 via HIF-1α in Mitochondrial COX4-1 Deficiency

Abstract

Cytochrome-c-oxidase (COX) subunit 4 (COX4) plays important roles in the function, assembly and regulation of COX (mitochondrial respiratory complex 4), the terminal electron acceptor of the oxidative phosphorylation (OXPHOS) system. The principal COX4 isoform, COX4-1, is expressed in all tissues, whereas COX4-2 is mainly expressed in the lungs, or under hypoxia and other stress conditions. We have previously described a patient with a COX4-1 defect with a relatively mild presentation compared to other primary COX deficiencies, and hypothesized that this could be the result of a compensatory upregulation of COX4-2. To this end, COX4-1 was downregulated by shRNAs in human foreskin fibroblasts (HFF) and compared to the patient’s cells. COX4-1, COX4-2 and HIF-1α were detected by immunocytochemistry. The mRNA transcripts of both COX4 isoforms and HIF-1 target genes were quantified by RT-qPCR. COX activity and OXPHOS function were measured by enzymatic and oxygen consumption assays, respectively. Pathways were analyzed by CEL-Seq2 and by RT-qPCR. We demonstrated elevated COX4-2 levels in the COX4-1-deficient cells, with a concomitant HIF-1α stabilization, nuclear localization and upregulation of the hypoxia and glycolysis pathways. We suggest that COX4-2 and HIF-1α are upregulated also in normoxia as a compensatory mechanism in COX4-1 deficiency.

Mitochondrial COX4-1 deficiency leads to impaired nuclear DNA damage response resulting in proliferation deficits and premature senescence

“…Altogether, our results suggest that the proliferation de cits in COX4-1-de cient cells is more likely attributed to senescence than apoptosis. This is also in accord with our previous observations [10] detecting increased glycolysis in these cells. Notably, metabolic shift, towards glycolysis occurs in senescence [19] 1.2 Nuclear DNA instability coincides with proliferation We previously showed that COX4-1-de cient cells display high levels of DSB relative to controls by performing both phospho-histone H2AX Ser139 (γH2AX) foci staining and neutral comet assay [11,12].…”

“…As transformation is much less e cient in primary cells such as HFF, we constitutively knocked-down COX4I1 in HEK 293 cell line (HEK293-shCOX4I1) instead. Knockdown e ciency, as well as the presence of DSB were both validated in the HEK293 cells before commencing, and the results were comparable to those we reported in HFF-shCOX4I1 cells [10] (supplementary gure S2). Subsequently, we co-transfected the mutant LacZα plasmids (dl-1&dl-2) into both HEK293-shCOX4I1 and HEK293-CV, overnight and analyzed the resulting products by PCR (Fig.…”

“…Conversely, oxidative stress associated with mitochondrial dysfunction have both been previously implied in the classical form of Fanconi anemia, in patients' cells harboring mutations in the FA pathway genes [13][14]. Mitochondrial dysfunction also compels FA cells towards glycolytic metabolism [15] and this is similarly to our observations in the COX4-1-de cient cells [ 10].…”

“…For experiments the cells were maintained in permissive medium without puromycin. Knockdown was veri ed by RT-qPCR as we have previously described [10].…”

“…We demonstrated the involvement of HIF-1α stabilization and nuclear translocation, that was followed by COX4 isoform switch under normoxic conditions. We thus hypothesized that the patient's relatively mild phenotype could be related to the isoform switch which preserving COX partial activity [10].…”